1. General Information
This specification contains nucleotide and amino acid sequence information prepared using PatentIn Version 3.3, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, <210>3, etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term “SEQ ID NO:”, followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>1).
The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
Each embodiment described herein is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts:    1. Sambrook, Fritsch & Maniatis, whole of Vols I, II, and III;    2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;    3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp 1-22; Atkinson et al., pp 35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151;    4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;    5. Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of text;    6. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text;    7. Perbal, B., A Practical Guide to Molecular Cloning (1984);    8. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series;    9. J. F. Ramalho Ortigão, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany);    10. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342    11. Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154.    12. Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.    13. Wünsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Müler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart.    14. Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg.    15. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg.    16. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.    17. Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications).    18. McPherson et al., In: PCR A Practical Approach., IRL Press, Oxford University Press, Oxford, United Kingdom, 1991.    19. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual (D. Burke et al., eds) Cold Spring Harbor Press, New York, 2000 (see whole of text).    20. Guide to Yeast Genetics and Molecular Biology. In: Methods in Enzymology Series, Vol. 194 (C. Guthrie and G. R. Fink eds) Academic Press, London, 1991 2000 (see whole of text).
2. Description of the Related Art
Peptide Therapeutics
As a response to the increasing demand for new lead compounds and new target identification and validation reagents, the pharmaceutical industry has increased its screening of various sources for new lead compounds having a unique activity or specificity in therapeutic applications, such as, for example, in the treatment of neoplastic disorders, infection, modulating immunity, autoimmunity, fertility, etc.
It is known that proteins bind to other proteins, antigens, antibodies, nucleic acids, and carbohydrates. Such binding enables the protein to effect changes in a wide variety of biological processes in all living organisms. As a consequence, proteins represent an important source of natural modulators of phenotype. Accordingly, peptides that modulate the binding activity of a protein represent attractive lead compounds (drug candidates) in primary or secondary drug screening. For example, the formation of a target biological interaction that has a deleterious effect (eg. replication of a pathogen or of a cancer cell), can be assayed to identify lead compounds that antagonize the biological interaction.
Antibodies represent the fastest growing class of approved drugs in this area, however they require complex and expensive synthesis and are difficult to deliver via non-injectable routes. In contrast, large peptides can often be made synthetically and are increasingly being delivered by buccal, intranasal or intranasal routes as alternatives to injection. Furthermore, intracellular delivery of peptides is also now possible in vivo using protein transduction domains. These advances make peptide-based therapeutics an attractive alternative to antibody-based therapeutics.
Existing drawbacks associated with peptide-based therapeutics include their low affinity, high turnover in vivo and difficulties in their isolation compared to small molecules. For example, peptides that target protein interaction interfaces which may be large and relatively featureless are generally more difficult to produce and isolate when compared to small molecule inhibitors of enzyme-active sites that generally form small complex pockets. Accordingly, it is not facile to identify peptides that address these problems.
For example, random peptide (synthetic mimetic or mimotope) libraries can be produced using short random oligonucleotides produced by synthetic combinatorial chemistry, cloned into an appropriate vehicle for expression, and the encoded peptide screened using one of a variety of approaches. However, the ability to isolate active peptides from random fragment libraries can be highly variable with low affinity interactions occurring between many of the peptide-binding partners and very low hit-rates for biologically active peptides. Moreover, the expressed peptides often show little or none of the secondary or tertiary structure required for efficient binding activity, and/or are unstable. This is not surprising, considering that biological molecules appear to recognize shape and charge rather than primary sequence (Yang and Honig J. Mol. Biol. 301(3), 691-711 2000) and that such random peptides are generally too small to comprise a protein domain or to form the secondary structure of a protein domain. Moreover even the largest peptide libraries to have been produced do not contain sufficient complexities to exhaustively cover all of the possible combinations of the 20 amino acids, for peptides of more than approximately a dozen residues. The relatively unstructured ‘linear’ nature of many artificial peptides derived from random amino acid sequences also leads to their more rapid degradation and clearance following administration to a subject in vivo, thereby reducing their appeal as therapeutic agents.
In contrast, natural protein folds or subdomains are understood in the art to mean independently folding peptide structures (e.g., a 19-residue fragment from the C-loop of the fourth epidermal growth factor-like domain of thrombomodulin as been described by Alder et al, J. Biol. Chem., 270: 23366-23372, 1995). These constrained structures provide thermodynamic advantages to bind other protein surfaces through limiting the entropic cost of binding. Moreover, structured folds can be less susceptible to proteolysis than unstructured linear peptides, increasing their biological stability.
To enhance the probability of obtaining useful bioactive peptides or proteins from random peptide libraries, peptides have previously been constrained within scaffold structures, eg., thioredoxin (Trx) loop (Blum et al. Proc. Natl. Acad. Sci. USA, 97, 2241-2246, 2000) or catalytically inactive staphylococcal nuclease (Norman et al, Science, 285, 591-595, 1999), to enhance their stability. Constraint of peptides within such structures has been shown, in some cases, to enhance the affinity of the interaction between the expressed peptides and its target, presumably by limiting the degrees of conformational freedom of the peptide, and thereby minimizing the entropic cost of binding.
Recently, peptide mimotopes of less than about 50 amino acids in length have been described that are capable of forming protein domains by virtue of assuming conformations sufficient for binding to a target protein or target nucleic acid (“Phylomer™ peptides”, Phylogica, Perth, western Australia, Australia) e.g., International Patent Application No. PCT/AU00/00414 and US Patent Publication No. 2003-0215846 A1. Such Phylomer™ peptides show promise in overcoming the existing drawbacks associated with peptide therapeutics. The conformation(s) of such Phylomer™ peptides is a product of secondary and/or tertiary structural features and, by virtue of the peptide binding to its target protein or protein interaction interface is compatible with, albeit not necessarily iterative of, the target protein(s) or target protein interaction interface. Such secondary structural features may suggest that Phylomer™ peptides, on average, have higher substrate affinities and longer half-lives than more conventional random peptides. On the other hand, Phylomer™ peptides may also provide production and delivery advantages compared to antibody-based therapies by virtue of their small size. Additionally, because Phylomer™ peptides are derived from libraries comprising mixtures of small genome fragments from evolutionarily-diverse bacteria and eukaryotes having small albeit well-characterized genomes, they can be screened in silico to select against those peptides sequences that are likely, because of their known strucure or function, to produce adverse reactions in recipient mammals, including humans. Notwithstanding the need for empirical testing of therapeutic products, this “safety” feature of Phylomer™ peptides provides a significant potential advantage over peptides derived from mammals, including antibodies.
Neuronal Disorders Involving Neuronal Cell Death
Neuronal disorders such as migraine, stroke, traumatic brain injury, epilepsy and neurodegenerative disorders including Huntington's Disease (HD), Parkinson's Disease (PD), Alzheimer's Disease (AD) and Amyotrophic Lateral Sclerosis (ALS) are major causes of morbidity and disability arising from long term brain injury. These effects generally involve apoptosis and/or necrosis of neurons, possibly involving diverse pathways including oxidative stress.
As used herein, the term “stroke” includes any ischemic disorder e.g., a peripheral vascular disorder, a venous thrombosis, a pulmonary embolus, a myocardial infarction, a transient ischemic attack, lung ischemia, unstable angina, a reversible ischemic neurological deficit, adjunct thromolytic activity, excessive clotting conditions, reperfusion injury, sickle cell anemia, a stroke disorder or an iatrogenically induced ischemic period such as angioplasty, or cerebral ischemia.
Glutamate Excitotoxicity
Increased extracellular levels of the neurotransmitter glutamate cause neuronal cell death via excitotoxicity. An accumulation of extracellular glutamate over-stimulates NMDA and AMPA receptors resulting in an influx of extracellular calcium and sodium ions and the release of bound calcium from intracellular stores. The increase in intracellular calcium initiates a range of cell damaging events involving phospholipases, proteases, phosphatases, kinases and nitric oxide synthase, as well as the activation of the pro-apoptotic transcription factor c-Jun.
Involvement of the AP-1 Signaling Pathway in Neuronal Function
Various types of evidence indicate that c-Jun N-Terminal Kinase (JNK or SAPK) is involved in neuronal cell death during or following ischemia, via activation of c-Jun (a component of the AP-1 complex) in an analogous way to the known activation of this stress kinase response in other forms of ischemia such as coronary heart disease or in organ or blood vessel reperfusion injury.
Components of the AP-1 pathway associate with scaffold proteins that modulate their activities and cellular localization. JNK activity is controlled by a cascade of protein kinases and by protein phosphatases, including dual-specificity MAPK phosphatases. For example, the JNK-interacting protein-1 (JIP-1) scaffold protein specifically binds JNK, MAPK kinase 4 (MKK4) and MAPK kinase 7 (MKK7), and members of the mixed lineage kinase (MLK) family, and regulates INK activation in neurons. Distinct regions within the N termini of MKK7 and the MLK family member dual leucine zipper kinase (DLK) mediate their binding to JIP-1. JNK binds to c-Jun, and this appears to be required for efficient c-Jun phosphorylation.
Several members of the death-related AP-1 pathway acting upstream of JNK have been defined. The most distal of these are the Rho small GTPase family members Rac1 and Cdc42. Over expression of constitutively active forms of Rac1 (i.e., Rac1V12) and Cdc42 (i.e., Cdc42V12) leads to activation of the AP-1 pathway and to death of Jurkat T lymphocytes, PC12 cells, and sympathetic neurons. Conversely, over expression of dominant-negative mutants of Cdc42 (i.e., Cdc42N17) and Rac1 (i.e., Rac1N17) in sympathetic neurons prevents elevation of c-Jun and death evoked by nerve growth factor (NGF) withdrawal (Bazenet et al., Proc. Natl. Acad. Sci. USA 95, 3984-3989, 1998; Chuang et al., Mol. Biol. Cell 8, 1687-1698, 1997). Over expression of the dominant negative mutant Rac1N17 also reverses the induction of death by Cdc42V12, whereas Cdc42N17 has no effect on Rac-1V12-induced death, suggesting that Cdc42 lies upstream of Rac1 (Bazenet et al., Proc. Natl. Acad. Sci. USA 95, 3984-3989, 1998). Similar approaches have indicated that mitogen-activated protein kinase kinases 4 and 7 (MKK4 and MKK7) lie downstream of Cdc42 and Rac1 and directly upstream of the JNKs (Foltz et al., J. Biol. Chem. 273, 9344-9351, 1998; Holland et al., J. Biol. Chem. 272, 24994-24998, 1997; Mazars et al., Oncogene 19, 1277-1287, 2000; Vacratsis et al., J. Biol. Chem. 275, 27893-27900, 2000; Xia et al., Science 270, 1326-1331, 1995; Yamauchi et al., J. Biol. Chem. 274, 1957-1965, 1999). Studies using constitutively active and dominant-negative constructs have also implicated apoptosis signal-regulating kinase 1 (ASK1) as an additional participant in the pathway that lies between Cdc42 and the downstream MKKs and JNKs (Kanamoto et al., Mol. Cell. Biol. 20, 196-204, 2000).
MLKs have been shown to function as MKK kinases and lead to activation of JNKs via activation of MKKs (Bock et al., J. Biol. Chem. 275, 14231-1424, 2000; Cuenda et al., Biochem. J. 333, 11-159, 1998; Hirai et al., J. Biol. Chem. 272, 15167-15173, 1997; Merritt et al., J. Biol. Chem. 274, 10195-10202, 1999; Rana et al., J. Biol. Chem. 271, 19025-19028, 1996; Tibbles et al., EMBO J. 15, 7026-7035, 1996; Vacratsis et al., J. Biol. Chem. 275, 27893-27900, 2000). Members of the family include MLK1, MLK2 (also called MST), MLK3 (also called SPRK or PTK1), dual leucine zipper kinase (DLK; also called MUK or ZPK), and leucine zipper-bearing kinase (LZK). Constitutively active mutants of Rac1 and Cdc42 have been found to bind to and to modulate the activities of MLK2 and -3, and co-expression of MLK3 and activated Cdc42 leads to enhanced MLK3 activation.
In animal models of ischemia or migraine, stroke, apoptotic neurons have enhanced phosphorylation of the transcription factor c-Jun by JNK. Additionally, neuronal c-Jun levels are elevated in response to trophic factor withdrawal, and dominant-negative forms of this transcription factor are at least partially-protective against neuronal cell death evoked by selective activation of JNKs (Filers et al., J. Neurosci. 18, 1713-1724, 1998; Ham et al., Neuron 14, 927-939).
The transcriptional activating activity of c-Jun is regulated at the post-translational level by its phosphorylation by JNK (SAPK) at two residues within the amino-terminal trans-activation domain, serines 63 and 73, in response to a variety of cellular stresses. Phosphorylation of these two residues is critical for the transcriptional activating activity of c-Jun, since mutation of them markedly decreases this activity. JNKs (SAPKs) readily phosphorylate c-Jun at Ser 63/73, and at a rate that is about 10 times faster than ERK-1 and ERK-2. The JNKs (SAPKs) account for the majority of c-Jun trans-activation domain (Ser 63/73) kinase activity after reperfusion, suggesting that they trigger part of the kidney's very early genetic response to ischemia by enhancing the transcriptional activating activity of c-Jun. Since induction of c-Jun is auto-regulated, it is likely that activation of the JNKs (SAPKs) is, at least in part, responsible for the induction of c-Jun following myocardial or renal ischemia.
The role of JNKs (SAPKs) in the control of gene expression during and/or following ischemia extends well beyond the regulation of c-Jun by INK. It is known that AP-1 comprises complexes of c-Jun with parters such as c-Fos or ATF-2 (a member of the CREB family). When complexed with c-Fos, the dimer is targeted to promoters, such as that of the collagenase gene, containing canonical AP-1 elements. When complexed with ATF-2, however, the dimer appears to prefer CRE sequences, and AP-1 variants such as that contained in the c-Jun promoter which controls induction of c-Jun in response to a variety of stimuli. After ischemia and reperfusion, ATF-2 and c-Jun are targeted as a heterodimer to both ATF/CRE motifs and the Jun2 TRE within the c-Jun promoter. This suggests that, following reperfusion of ischemic tissue, the JNKs (SAPKs) target ATF-2/c-Jun heterodimers to various promoters, including the c-Jun promoter, and enhance transcriptional activating activity of both components of the c-Jun/ATF-2 dimer. This may provide a potent mechanism for the induction of a large number of genes regulated by promoters containing ATF/CRE sites or AP-1 variants to which the heterodimer binds.
Dimerization of c-Jun also leads to apoptosis in neurons in response to ischemia (Tong et al., J. Neurochem 71, 447-459, 1998; Ham et al., Biochem. Pharmacol. 60, 1015-1021, 2000).
A homodimer of c-Jun is also known to activate the c-Jun transcription factor via binding to the transcriptional regulatory element (TRE) in the c-Jun promoter.
As used herein unless specifically stated otherwise or the context requires otherwise, the term “c-Jun dimerization” shall be taken to include homo-dimerization of c-Jun monomers and the partnering of c-Jun with another peptide or polypeptide e.g., JNK, c-Fos, ATF-2. Similarly, unless specifically stated otherwise or the context requires otherwise, the term “c-Jun dimer” shall be taken to include homo-dimer of c-Jun monomers and a heterodimer of c-Jun with another peptide or polypeptide e.g., c-Fos, ATF-2, including transient complexes such as those between the INK kinase and its substrate c-Jun.
Treatment of Neuronal Cell Death
Currently, there is no effective clinical agent that inhibits the delayed neuronal cell death associated with such neuronal dysfunction. For example, drugs such as Activase (genetically engineered tissue plasminogen activator; Genentech), Abciximab (a platelet inhibitor; Centocor), and Ancrod (fibrinogenolytic) have had limited success, even if administered soon after the stroke occurs. These agents offer no clinical benefit if administered later than the period immediately following the stroke and unfortunately many patients present to a hospital after this window of opportunity. Even alternative approaches that target glutamate receptors to prevent glutamate excitotoxicity causing neuronal damage have shown no significant or consistent improvements in patient outcome, most likely due to the need to target these events early.